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Abstract

Background

Endothelial cells play an important role in the delivery of cells to the inflammation
site, chemotaxis, cell adhesion and extravasation. Implantation of a foreign material
into the human body determines inflammatory and repair reactions, involving different
cell types with a plethora of released chemical mediators. The evaluation of the interaction
of endothelial cells and implanted materials must take into account other parameters
in addition to the analysis of maintenance of cell viability.

Methods

In the present investigation, we examined the behavior of human umbilical vein endothelial
cells (HUVECs) harvested on titanium (Ti), using histological and immunohistochemical
methods. The cells, after two passages, were seeded in a standard density on commercially
plate-shaped titanium pieces, and maintained for 1, 7 or 14 days.

Results

After 14 days, we could observe a confluent monolayer of endothelial cells (ECs) on
the titanium surface. Upon one-day Ti/cell contact the expression of fibronectin was
predominantly cytoplasmatic and stronger than on the control surface. It was observed
strong and uniform cell expression along the time of α5β1 integrin on the cells in
contact with titanium.

Conclusion

The attachment of ECs on titanium was found to be related to cellular-derived fibronectin
and the binding to its specific receptor, the α5β1 integrin. It was observed that
titanium effectively serves as a suitable substrate for endothelial cell attachment,
growth and proliferation. However, upon a 7-day contact with Ti, the Weibel-Palade
bodies appeared to be not fully processed and exhibited an anomalous morphology, with
corresponding alterations of PECAM-1 localization.

Background

Since the discovery of endothelial-derived relaxing factor (EDRF) by Furchgott & Zawadzki
[1], in 1980, endothelial cells (ECs) have been recognized to be involved in vascular
homeostasis, angiogenesis and repair of injured tissues. ECs play an important role
in the trafficking of cells from bloodstream towards an inflammatory site, chemotaxis,
cell adhesion and extravasation [2]. Factors released by ECs mediate the control of vascular tonus, thrombogenesis and
fibrinolysis, and platelet activities [3]. Besides, by interacting with cytokines and leukocytes, ECs orchestrate the inflammatory
process [4], a fact involved with the complex phenomena observed at the host implant interface.
ECs produce and store the haemosthatic protein von Willebrand factor (vWf) into granules
named Weibel Palade bodies (WPBs), that are secretory organelles. They thus provide
a readily releasable pool of extracellular VWF as well as placing P-selectin on the
plasma membrane whereby it can recruit leukocytes and thus play a role in the initiation
of inflammation [5].

Implantation of a foreign material into the intimity of human tissues triggers a typical
inflammatory response followed by tissue repair. After implanted, the material will
determine the clinical outcome and will have an influence on the implantation bed,
triggering cellular and non-cellular responses [6]. Metals and alloys are the most common materials used as surgical implants in order
to replace mineralised structures [7,8]. In particular, titanium alloys show properties which render them suitable substrates
for surgical implant [6,8]. Moreover, the high degree of biocompatibility of titanium and its alloys is intimately
related to the passively formed oxide film on the metallic surface [9,10].

Noteworthy, the evaluation of the interaction of cells and implanted materials must
take into account other parameters in addition to the analysis of maintenance of cell
viability. Indeed, the interaction of implants with host cells, and in particular
with endothelial cells, might cause activation of adhesion molecules culminating with
cytokine generation [2]. In fact, the degree of expression of adhesion molecules on the surface of human
ECs depends on the response of the cells against the implanted material [11].

PECAM-1 is a cell-cell junction molecule that establishes homophilic binding between
neighboring ECs [12]. PECAM-1 interacts with the underlying cytoskeleton and regulates F-actin assembly
at the cell periphery in association with changes in cell shape and spreading [13]. The mechanism of endothelial cell adhesion to substrates involves integrins expression,
thence connecting extracellular matrix (ECM) with the cytoskeleton [14,15]. Integrins are also considered to be the main receptors of ECM proteins, such as
fibronectin, laminin, collagens, and vitronectin. Altogether, these proteins constitute
the main mediators of cell-ECM adhesion [14].

There is evidence that upon binding to an ECM protein (e.g. fibronectin), a number
of integrins mediate cellular signaling and functions. It was shown that α5β1 integrin,
a receptor for both fibronectin (FN) and vitronectin (VN), and αvβ3 integrin, a VN
receptor, both play a role in angiogenesis [16]. Therefore, the success of vasculogenesis and angiogenesis depends on FN [17,18] and its main receptor, the α5β1 integrin [19]. Upon wound repair, angiogenetic mechanisms are called into play leading to generation
of new capillary blood vessels [20]. Accordingly, angiogenesis is of pivotal importance during the initial healing process,
and thus the characterization of the cellular responses involved in angiogenesis and
bone formation adjacent to the implants is critical to understanding and promoting
implant biocompatibility and improving stable fixation of implants [21].

Tissue repair around an implanted piece of Ti depends crucially on osseous integration
and angiogenesis. Though a huge deal of information exist about bone modifications
in this situation, the interaction of this metal with endothelial cells is not completely
understood. Being so, in this study we investigated the behavior of ECs in culture
on Ti plates and assessed the protein expression and cell adhesion, in an attempt
to better understand the reasons why Ti-made implant materials achieve successful
clinical application.

Materials and methods

The experimental design was approved by the Ethics Committee, Faculty of Medicine,
University of Münster. The material analyzed in this study was commercially available,
plate-shaped pieces of pure titanium (Ti). As control surfaces, we used round plastic
coverslips (Thermanox®, Nunc, USA) coated with gelatin (Sigma, USA).

Cultures of human umbilical vein endothelial cells (HUVECs)

The endothelial cells were isolated from umbilical cord veins essentially as described
by Marin et al. [22], according to the method of Jaffe et al. [23]. Cells were pooled and established as primary cultures seeded on 0,5% gelatin-coated
(gelatin 2% solution) tissue culture dishes in medium 199 enriched with 20% heat-inactivated
fetal bovine serum, 5 μg/ml amphotericin B, 200 U/ml penicillin, 200 μg/ml streptomycin,
1% endothelial cell growth supplement, and 0,1% heparin. Cultures were carried out
at 37°C in a humidified atmosphere with 5% CO2; the culture medium was changed every other day. The cultures were serially passaged
by incubating confluent cells in 0.05% trypsin/0.02% EDTA solution and replating them
at a 1:2 ratio. Second passage cells were taken for the experiments after being identified
as endothelial cells by staining with a panel of endothelial-specific antibodies (anti-vWF,
anti-CD-31 [PECAM-1]) and by a negative staining to anti-α-smooth muscle actin.

Contact assays

Detachment of cells was performed by trypsinisation during 1 min; the reaction was
stopped by dilution with enriched medium and the resulting suspensions were centrifuged
(1,200 rpm for 3 min). The pellet was resuspended in growth medium. Cell numbers were
determined with a cell counter. Titanium plates (1 cm2) were placed onto the bottoms of 24-well plates, and the cells were seeded in every
well at a density of 8 × 104 cells/cm2. As control surfaces, we used round pieces (plastic coverslips) with the same dimensions,
coated with gelatin and seeded with the same concentration of cells. The experiments
were carried out during 1, 7 or 14 days and the media were changed every other day.

Immunohistochemistry

At the end of every culture period, both cell substrates were processed with a fluorescent
staining method and studied with a confocal microscope. For this purpose, the substrates
inside the wells were rinsed with PBS and fixed in methanol for 20 minutes at -4°C.
Non-specific sites were then blocked by incubation during 15 minutes at room temperature
with Tris-buffered saline/Tween 20® (TBST) containing 0.5% bovine serum albumin. Afterwards, the substrates were incubated
with the primary antibodies during 1 h at 37°C, rinsed three times with TBST and then
incubated with the correspondent alexa fluor 488 second antibody for 1 h at 37°C.
Negative controls were prepared using incubations with primary antibodies-free saline.
Images of the stained probes were captured using a confocal microscope (Zeiss Axiovert).

Scanning electron microscopy

The titanium plates were fixed with 2.5% glutaraldehyde and then dehydrated using
a graded ethanol series. The process was completed by critical point drying using
CO2 and a thin layer of gold was sputter-coated onto the plates prior to examination.
The images of the surfaces were captured using a Philips PSEM500× microscope.

Cell countings

After the corresponding culture periods, titanium and control pieces were rinsed with
PBS to eliminate unattached cells. The adherent cells were removed by incubation with
trypsin/EDTA for 2 minutes at 37°C. Trypsin action was stopped by dilution with complete
growth medium at room temperature and 100 μl-samples of the resulting cell suspensions
were counted using a cell counter (Casy®1, Schärfe System, Germany). Results were expressed as the number of adherent cells
per sample of titanium or control and were analyzed by one-way analysis of variance
and the Tukey-Kramer multiple comparisons test. A 2.01 version GraphPad InStat™ software
was used for this purpose. When appropriate, the Student's t test was also used.

Cell morphology

The morphological observations indicated that the cells seeded on titanium seem to
conform to the material surface and to be flat, elongated and oriented along the titanium
grooves (Figure 2A). After 7 days we could observe the presence of increased number of cells; after
14 days there was a confluent and dense cell layer (Figure 2B). Cells over the control surface maintained their morphological patterns without
any significant volume variation or other modifications.

Figure 2.Scanning electron microscopy image of endothelial cells attached to the titanium plate
after 1 day of culture (A) and after 14 days (B) (1250 ×).

Titanium assays and immunohistochemistry

HUVECs adhered to the surface, spread and proliferated and within 24 hours started
forming a subconfluent monolayer. On titanium and on control surfaces it could be
noticed that the cells proliferated and reached confluence, throughout the experimental
period.

Stainings with antibodies anti- PECAM-1 and anti-vWf were performed to confirm the
conservation of the endothelial characteristics. There was a mild up to strong expression
of PECAM-1 by the cells and no expression was evidenced on the cellular processes
after 7 days (Figures 3A, B and 3C). Conversely, on the control surfaces the CD-31 expression did not vary during the
studied periods (Figures 3D, E and 3F). Cells tested for vWf showed the presence of this factor on titanium and on control
surfaces, but on titanium after 7 and 14 days we could not observe well-defined granules
in the cells and after 7 days they are distributed on the perinuclear region (Figure
4B and 4C). Conversely, on control surfaces the cells presented well-defined vWf granules uniformly
distributed in the cells in all studied periods (Figures 4D, E and 4F).

Figure 3.Images of the immunohistochemical assays: PECAM-1 on endothelial cells attached to
titanium after (A) 24 hours, (B) 7 days and (C) 14 days, and to control surface after
(D) 24 hours, (E) 7 days and (F) 14 days.

Figure 4.Images of the immunohistochemical assays: von Willebrand factor on endothelial cells
attached to titanium after (A) 24 hours, (B) 7 days and (C) 14 days, and to control
surface after (D) 24 hours, (E) 7 days and (F) 14 days.

The cells were also studied for the presence of extracellular matrix proteins, fibronectin
and vitronectin. The results on both surfaces showed a strong positive reaction for
fibronectin with a progressively intensity increase. Upon one-day Ti/cell contact
the expression of fibronectin was predominantly cytoplasmatic and stronger than on
the control surface (Figure 5A and 5D). After 7 and 14 days we observed that fibronectin was predominantly extracellular
on both surfaces (Figures 5B, C, E and 5F). In contrast, no positive responses were obtained with the specific anti-vitronectin
antibody.

Figure 5.Images of the immunohistochemical assays: fibronectin on endothelial cells on titanium
after (A) 24 hours, (B) 7 days and (C) 14 days and on control surface after (D) 24
hours, (E) 7 days and (F) 14 days.

Assays for VE-cadherin expression resulted negative, both on control and titanium
surfaces for any experimental period studied.

The assays for integrins α5β1 (Figure 6) and α(v)β3 (Figure 7) revealed strong and uniform cell expression along the time, with a stronger reaction
for α5β1 on the cells in contact with titanium or control surfaces.

Figure 6.Images of the immunohistochemical assays: α5β1 integrin expression on endothelial
cells attached to titanium after (A) 24 hours, (B) 7 days and (C) 14 days and to control
surface (D).

Figure 7.Images of the immunohistochemical assays: α(v)β3 integrin expression on endothelial
cells attached to titanium after (A) 24 hours, (B) 7 days and (C) 14 days and to control
surface (D).

Similar testings were done for vinculin; the staining was positive and did not significantly
vary along the time. The subcellular distribution of this protein, along the Ti/cell
contact time, was similar to that of the α5β1 integrin found after 7 days.

Cell countings

Figure 8 shows the number of cells attached to titanium and control surfaces after the different
growing periods (1, 7 or 14 days). Overall, there was an increasing number of cells
adhered to the substrates along the time, without significant difference between titanium
and control surface.

Figure 8.Attachment of HUVECs on titanium and on control surfaces after the studied periods. Values are mean ± SEM.

Discussion

Besides requiring invasion by endothelial cells (ECs), angiogenesis depends upon localized
proteolytic modifications of the extracellular matrix (ECM) and/or a substrate to
which ECs can adhere, migrate upon, proliferate within, and eventually differentiate
into a mature EC phenotype. The physical characteristics as well as the composition
of the material must be suitable for cell adherence, because focal adhesions can only
be formed and maintained when the material sustains cell adhesion. The cellular response
to a foreign material is dictated largely by the surface properties of the material
to which the cells contact. A better understanding of the role of occurring matrix
in the various steps of angiogenesis would conceivably contribute to the intelligent
design of tissue-engineered constructs where angiogenesis is critical for tissue repair
and restoration [24].

Results from van Kooten et al. [25] showed that HUVECs adhere to metal surfaces and start forming a subconfluent monolayer
within 3 days, and that focal contacts are present after 3 days of adhesion, with
the cells still displaying their endothelial phenotype. It has been demonstrated that
various specific proteins adsorb onto the scaffolds used for culture from serum-containing
medium in vitro, and that cells use specific integrin receptors to bind to these proteins [26].

Tissue repair around an implanted piece of Ti depends crucially on osseous integration
and angiogenesis. Though a huge deal of information exist about bone modifications
in this situation, the interaction of endothelial cells with Ti is not completely
understood. A permanent state of oxidative stress appears to exist in endothelial
cells grown in direct contact with Ti surfaces [27]. These authors showed that HUVECs adhere to Ti in vitro and a complete coverage of an EC layer was obtained without any coating or surface
treatment.

Cell adhesion to implants in vivo and to culture surfaces in vitro is typically dependent upon surface-adsorbed fibronectin and vitronectin [28]. After 1 day on titanium (see Fig. 5) it could be observed a strong cytoplasmatic pool of fibronectin on ECs and a growing
extracellular mesh. Conversely, on control surfaces that fibronectin pool was not
observed. After 7 days this protein was found over both surfaces with a more dense
mesh on the control surfaces. The cells produced and released fibronectin in order
to use this protein as a substrate for attachment, with a stronger presence in the
cells after 1 day on titanium. On control surfaces the initial attachment was most
conceivably based on the gelatin coat. Although it has been stated that over time
the adhesion may be progressively more dependent on vitronectin in view of the larger
amounts, or the preferential binding to this molecule [29,30], our data do not support this view, since our EC vitronectin stainings failed to
demonstrate any positive answer either on titanium or control surfaces. Our findings
showed that ECs produced and eliminated endogenous fibronectin, using this protein
to attach to the surface through integrins (namely, αvβ1 and αvβ3) which are expressed
when ECs were seeded on titanium. The observed expression of αvβ3 was weaker in comparison
to that of α5β1 (Figs. 6 and 7).

The EC morphology on titanium plates was not altered. However, WPBs were not well-defined
after 7 and 14 days and they were distributed on the perinuclear region. Some authors
report that only fully processed, substantially polymerized and functionally mature
vWf is stored in the WPBs, together with its propeptide [31]. Interestingly, it is established that HUVECs poses two different populations of
WPBs, that are differentiated at cellular level by their distribution, the newly formed
ones being immature and located in the perinuclear region [5]. Thus it is conceivable to infer that on titanium, after 7 and 14 days, the WPB on
HUVECs were immature in nature as judged by their distribution (Fig. 4) whereas the mature granules were secreted. On the other hand, the presence and the
distribution of the WPBs on HUVECs on control surfaces were uniform in all studied
periods.

Since PECAM-1-mediated pathway could be involved in the observed differences in cell
behavior, the expression level of PECAM-1 on titanium was analyzed. CD-31 (PECAM-1)
was expressed mildly up to strongly by the cells on Ti, but we could not observe the
expressed CD-31 on the cellular processes after 7 days (Fig. 3). Since this was not observed with control surfaces, those cell contact alterations
might well be due to an effect of titanium on HUVECs.

Our results did not show the presence of VE-cadherin within junctions in all periods
studied neither in contact with titanium nor with the control surface. This could
be an evidence of a lack of some stimulus (i) for this molecule to trigger its participation
on the junctions.

Conclusion

In conclusion, our data indicated that the attachment of ECs on titanium could be
related to cellular-derived fibronectin and the binding to its specific receptor,
the α5β1 integrin. It was observed that titanium effectively serves as a suitable
substrate for endothelial cell attachment, growth and proliferation in the initial
phase. It is suggested to describe this feature of titanium if not angiogenic then
at least angio-conductive. However, upon a 7-day contact with Ti the Weibel-Palade
bodies were found to be not fully processed and with altered morphology, which corresponding
alterations of PECAM-1 localization. In fact that the successful Ti devices implantation
coexists with the microscopical subclinical adverse effects is, at the present, unresolved.

Authors' contributions

ACB–F performed all experiments under the supervision of JK. ACB–F drafted the manuscript
and was advised by RMO–F and WTdL. All authors read and approved the final manuscript.

Acknowledgements

We thank Mrs. Schuette, Mrs. Hartmann and Mr. Huda for their useful and informative
technical assistance.